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First published online November 7, 2008
doi: 10.1242/10.1242/dev.025080

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miRNAs are essential for survival and differentiation of newborn neurons but not for expansion of neural progenitors during early neurogenesis in the mouse embryonic neocortex

Davide De Pietri Tonelli^1,*,, Jeremy N. Pulvers¹, Christiane Haffner¹, Elizabeth P. Murchison², Gregory J. Hannon² and Wieland B. Huttner^1,

¹ Max-Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
² Cold Spring Harbor Laboratory, Watson School of Biological Sciences and Howard Hughes Medical Institute, Cold Spring Harbor, NY 11724, USA.

View larger version (55K): [in this window] [in a new window]   Fig. 1. Conditional ablation of Dicer in neural progenitors of the dorsal telencephalon during mouse embryonic development results in a smaller cortex. (A) Intrinsic EGFP fluorescence (white) with (a) and without (b) DAPI staining (blue) in a 10-µm coronal cryosection through the brain of an E13.5 mouse embryo obtained from crossing an Emx1Cre/wt mouse with a Z/EG reporter mouse. Note the specific expression of EGFP in the dorsal telecephalon. Mb, midbrain. The dashed box in panel a indicates the region shown at higher magnification in panel b; this region was chosen for subsequent analyses of high-magnification images (20x objective). (B-E) In situ hybridization on 10-µm cryosections through the heads of E10.5-12.5 control (Emx1Cre/wt Dicerflox/wt) and conditional Dicer knockout (Dicer KO, Emx1Cre/wt Dicerflox/flox) littermate embryos, using LNA antisense probes for miR-9 (B,C) and miR-124 (D,E). (B,D) Low-magnification overviews. Brackets indicate the region of the dorsal telencephalon used for subsequent analyses; dotted lines and arrows indicate the ventral telencephalon that is unaffected by Emx1-Cre-mediated Dicer ablation. (C,E) Higher magnification of the E10.5 (E) and E11.5 (C) mesencephalon and of the E10.5-12.5 dorsal telencephalon. Note the lack of accumulation of mature miR-9 (B,C) and miR-124 (D,E) specifically in the dorsal telencephalon upon Dicer ablation. VZ, ventricular zone; BL, basal lamina; brackets in C,E indicate the pre-plate (E11.5) and cortical plate (E12.5). (F) Comparison of P0 brains of control (Emx1Cre/wt Dicerflox/wt, left) and conditional Dicer knockout (Dicer KO, Emx1Cre/wt Dicerflox/flox, right) littermate mice. (Top) Dissected brains. Note the reduced size of the cerebral cortex (Cx) and olfactory bulbs (OB) in the Dicer KO brains. Dashed lines indicate the location of the coronal cryosections shown in F' and F''. (F',F'') DAPI staining of 10 µm coronal cryosections. Note the reduced size of the cortex (Cx) and hippocampus (Hp) (which lacks its typical structure), but not midbrain (Mb), in the Dicer KO brains. Scale bars: 500 µm in F',F''; 250 µm in Aa,B,D; 100 µm in Ab; 50 µm in C,E.

View larger version (88K): [in this window] [in a new window]   Fig. 2. Reduced thickness of the neuronal layers, but not the progenitor layers, in the Dicer-ablated E13.5 dorsal telencephalon. Comparison of the dorsal telencephalon of control (Emx1Cre/wt Dicerflox/wt) and conditional Dicer knockout (Dicer KO, Emx1Cre/wt Dicerflox/flox) littermate mouse embryos. (A-C) β III-tubulin (Tuj1) immunofluorescence confocal microscopy (3-µm single optical sections) of 10-µm coronal cryosections at E12.5 (A, top), E13.5 (B, top) and E14.5 (C); the E12.5 and E13.5 cryosections were co-stained with DAPI (A,B, bottom). Note the similar thickness of the neuronal layers in the control and Dicer KO cortex at E12.5 (A, arrowheads), the reduced thickness in the E13.5 Dicer KO cortex (B, arrowheads), and the absence of a recognizable structure of the neuronal layers (NL) in the E14.5 Dicer KO cortex (C). IZ, intermediate zone; SP, subplate; CP, cortical plate; MZ, marginal zone; Ec, ectoderm. (D) Tbr1 immunofluorescence microscopy of 10-µm coronal cryosections at E13.5. Dotted lines, basal boundary of SVZ; NL, neuronal layers; dashed lines, basal lamina. (E) Quantification of the area of the progenitor layers (VZ plus SVZ, black bars) and of the neuronal layers (NL, white bars), as indicated by the dashed white lines in D. Data are the mean of two embryos; for each embryo, the sum of the respective area of nine cryosections along the rostrocaudal axis (one field per cryosection) was calculated; bars indicate the variation of the two embryos from the mean. **P<0.01. (F) DAPI staining of 70 µm coronal Vibratome-prepared sections at E13.5. The length of the ventricular surface between the two arrowheads was quantitated (see G). (G) Quantification of the length of the surface of the lateral ventricle. Data are the mean of two embryos; for each embryo, the sum of the length of the ventricular surface in each of 12 consecutive sections along the rostrocaudal axis was calculated; bars indicate the variation of the two embryos from the mean. Asterisks (A-C,D,F) indicate the ventricular lumen. Scale bars: 500 µm in F; 200 µm in A,B; 100 µm in D; 50 µm in C.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Reduced thickness of neuronal layers in the Dicer-ablated E13.5 dorsal telencephalon is not due to loss of apical and basal neurogenic progenitors. (A-H) Immunofluorescence microscopy of 10-µm coronal cryosections through the dorsal telencephalon of control [Emx1Cre/wt Dicerflox/wt (A,C) or Emx1Cre/wt Dicerflox/wt Tis21-GFP+/- (E,G)] and conditional Dicer knockout [Dicer KO, Emx1Cre/wt Dicerflox/flox (B,D) or Emx1Cre/wt Dicerflox/flox Tis21-GFP+/- (F,H)] E13.5 littermate mouse embryos, showing Pax6 (A-D), Tbr2 (E,F) and Tis21-GFP (G,H) staining. nl, neuronal layers; asterisks, ventricular lumen; dashed lines, boundaries of the SVZ; solid lines, basal lamina. Scale bars: 200 µm in A,B; 50 µm in C-H. (I,J) Quantification of Tbr2-positive (I) and Tis21-GFP-positive (J) nuclei in the VZ plus SVZ (as indicated by the upper dashed lines in E-H), each expressed as a percentage of total, DAPI-stained nuclei (not shown). Data are the mean of 28 fields counted per condition (two embryos, seven cryosections along the rostrocaudal axis per embryo, two fields per cryosection); bars indicate s.d.

View larger version (94K): [in this window] [in a new window]   Fig. 4. The reduced thickness of neuronal layers in the Dicer-ablated E13.5 dorsal telencephalon is not due to decreased cell cycle progression or division of apical and basal progenitors. (A,B,D,E,G,H) Immunofluorescence microscopy of 10-µm coronal cryosections through the dorsal telencephalon of control (Emx1Cre/wt Dicerflox/wt) and conditional Dicer knockout (Dicer KO, Emx1Cre/wt Dicerflox/flox) E12.5 (A,B), E13.5 (D,E) and E14.5 (G,H) littermate mouse embryos, showing phosphohistone H3 (PH3, red) and DAPI (white) staining. NL, neuronal layers; white arrows, mitotic apical progenitors; arrowheads, mitotic basal progenitors; yellow arrows, apoptotic nuclei. (C,F,I) Quantification of mitotic (phosphohistone H3-positive) apical and basal progenitors at E12.5 (C), E13.5 (F) and E14.5 (I). Data are the mean of 32 (C) or 28 (F,I) fields counted per condition from two embryos, eight (C) or seven (F,I) cryosections along the rostrocaudal axis per embryo, two fields per cryosection; bars indicate s.d. *P<0.05, **P<0.01. (J-L) Control (Emx1Cre/wt Dicerflox/wt; J,L black triangles) and conditional Dicer knockout (Dicer KO, Emx1Cre/wt Dicerflox/flox; K,L white diamonds) E12.5 littermate embryos were subjected to cumulative BrdU labeling in utero and analyzed after 4, 8 and 20 hours. (J,K) Triple immunofluorescence microscopy of 10-µm coronal cryosections through the dorsal telencephalon after 4 hours of cumulative BrdU labeling, showing BrdU (red), Tbr1 (green) and DAPI (blue) staining. NL, neuronal layers. (L) Quantification of BrdU-positive (BrdU+), Tbr1-negative nuclei in the VZ (immunostained as in J,K), expressed as percentage of DAPI-stained nuclei. Data are the mean of two embryos; for each embryo, the average for three fields along the rostrocaudal axis (one field per cryosection) was calculated; bars indicate the variation of the two embryos from the mean (and are often smaller than the size of the symbol used). Horizontal dashed lines indicate the growth fraction. Vertical dotted lines indicate TC-TS (control 10.5 hours, Dicer KO 11.0 hours); TS (length of S phase) was 2.4 hours for control and 3.4 hours for Dicer KO, and TC (total length of the cell cycle) was 12.9 hours for control and 14.4 hours for Dicer KO. Scale bars: 50 µm.

View larger version (85K): [in this window] [in a new window]   Fig. 5. Dicer ablation in the dorsal telencephalon causes progressive apoptosis starting at E12.5. (A-H) Immunofluorescence confocal microscopy (6-µm single optical sections) of 10-µm coronal cryosections through the dorsal telencephalon of control (Emx1Cre/wt Dicerflox/wt) and conditional Dicer knockout (Dicer KO, Emx1Cre/wt Dicerflox/flox) E10.5 (A,B), E12.5 (C,D), E13.5 (E,F) and E14.5 (G,H) littermate mouse embryos, showing βIII-tubulin (Tuj1, green), TUNEL (red) and DAPI (blue) staining. NL, neuronal layers. Scale bars: 50 µm. (I,J) Quantification of TUNEL-stained (TUNEL+) cells in the cortical wall. Data are the mean of two embryos; for each embryo, the average for three fields along the rostrocaudal axis (one field per cryosection) was calculated; bars indicate the variation of the two embryos from the mean. Note the interruption in the scale of the ordinate to accommodate the data for the E14.5 Dicer KO.

View larger version (87K): [in this window] [in a new window]   Fig. 6. Dicer ablation in the embryonic dorsal telencephalon results in reduced upper layer neurons, but not deep layer neurons, in the postnatal cortex. (A-F) Control (Emx1Cre/wt Dicerflox/wt; B,E) and conditional Dicer knockout (Dicer KO, Emx1Cre/wt Dicerflox/flox; C,F) mouse embryos were subjected to BrdU labeling in utero either at E12.5 plus E13.5 (B,C) or at E17.5 plus E18.5 (E,F), and the respective littermate pups were analyzed at P1, as outlined in A,D. (B,C,E,F) Triple immunofluorescence confocal microscopy (3-µm single optical sections) of 10-µm coronal cryosections through the caudal cortex stained for BrdU (red), Tbr1 (green) and Brn1 (green); DAPI staining in blue. Ventricular surface (VS), layers VI and V-I, and the pial surface (PS) are indicated on the left. Scale bar: 50 µm. (G,H) Quantification of Tbr1-positive (G) and Brn1-positive (H) nuclei (immunostained as in B,C,E,F) in the cortical wall (excluding the VZ; see the densely packed Brn1-positive layer in the control cortex at the bottom of the corresponding panel), each expressed as percentage of DAPI-stained nuclei. Data are the mean of three fields from independent experiments; one third of two of these fields is shown in B,E, and in C,F. Bars indicate s.d. (I,J) Quantification of BrdU-labeled (BrdU+) nuclei (immunostained as in B,C,E,F) in the cortical wall (defined as in G,H). Data are for the field of which one third is shown in B,C (I), and in E,F (J). BrdU-labeled nuclei were scored for immunoreactivity with Tbr1 (black), Brn1 (gray), or neither (white).

View larger version (48K): [in this window] [in a new window]   Fig. 7. Lack of Foxp2 expression in the postnatal Dicer-ablated cortex. (A-D) Immunofluorescence confocal microscopy of 10 µm coronal cryosections (A,B; 3-µm single optical sections) or 50 µm coronal Vibratome-produced sections (C,D; 6-µm single optical sections) through the cerebral cortex of control (Emx1Cre/wt Dicerflox/wt; A,C) and conditional Dicer knockout (Dicer KO, Emx1Cre/wt Dicerflox/flox; B,D) P0 (A,B) and P7 (C,D) littermate mice, showing DAPI (blue) and Foxp2 (red) staining. Note the almost complete absence of Foxp2-positive neurons in the cerebral cortex of P1 and P7 Dicer-ablated mice. Ventricular surface (VS), layers VI and V-I, and the pial surface (PS) are indicated on the left. Scale bars: 50 µm in A,B; 100 µm in C,D.

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